We are continuing our studies of a simple model system, NG108-15 neuroblastoma-glioma hybrid cells, to study the molecular basis of opioid tolerance/dependence. We have recently developed several new conceptual and experimental approaches, with which we have identified several molecules specifically affected by chronic opioid treatment of NG108-15 cells, and that therefore are likely to play some role in tolerance mechanisms of these cells. We propose to characterize these molecules structurally and to determine their functional significance. In the first approach, subtraction hybridization, mRNA is isolated from control and down-regulated cells, and cDNA-mRNA hybridization used to identify molecules specifically reduced or eliminated in the latter. Using this approach, we have recently identified, cloned and sequenced two closely-related mRNA's, NGD5A and NGD5B, that are down-regulated by chronic treatment with opioid but not muscarinic agonist, in a naloxone-reversible fashion. We propose a) to characterize this down-regulation with respect to time course and agonist/antagonist specificity; b) stably transfect and express NGD5 cDNA in neuro 2A cells, which do not contain opioid receptors; c) express NGD5A in E. coli, prepare antibodies to the protein, and test them for their effect, as well as determine the cellular location of the NGD5 product and follow changes in levels of the NGD5 product during chronic opioid treatment; d) prepare NGD5 genomic DNA in NG108-15 cells; e) compare NGD5 and any other down-regulated molecules identified by subtraction hybridization to opioid receptors that are purified directly from NG108-15 cells, using a detergent solubilization and affinity chromatography, cross-linking of 125I-beta-endorphin, and affinity and photoaffinity labelling. Finally, using a second novel approach, we will determine the effect of stable transfection of NGD5 antisense cDNA in NG108-15 cells on NGD5 mRNA levels, opioid binding and opioid inhibition of adenylate cyclase, and on chronic opioid effects. Our third approach is based on our recent purification of an opioid binding protein from bovine brain. Antibodies to this protein not only block binding to mu, delta and kappa opioid receptors in brain, but react with two distinct proteins in NG108-15 cells, of 58 and 39 kD; the 39 kD band is also down-regulated by chronic treatment of NG cells with opioid agonist. We will determine the location of these species on NG108-15 cells, follow the kinetics of 39 kD down-regulation, and purify the 58 and 39 kD proteins using an affinity column constructed from the antibody, followed by sequencing and cloning. Antisense cDNA to the sequences will be prepared, and tested for its effect on opioid binding, inhibition of cyclase, and chronic effects of opioids. Finally, we will compare the sequence and other structural characteristics of the 58 and 39 kD proteins to opioid receptors that are purified directly from NG108-15 cells, as in the first approach.